Identification of the principal neuropeptide MIP and its action pathway in larval settlement of the echiuran worm Urechis unicinctus

Background Larval settlement and metamorphosis represent critical events in the life history of marine benthic animals. Myoinhibitory peptide (MIP) plays a pivotal role in larval settlement of marine invertebrates. However, the molecular mechanisms of MIP involved in this process are not well understood. Results In this study, we evaluated the effects of thirteen MIP mature peptides on triggering the larval settlement of Urechis unicinctus (Xenopneusta, Urechidae), and determined that MIP2 was the principal neuropeptide. Transcriptomic analysis was employed to identify differentially expressed genes (DEGs) between the MIP2-treated larvae and normal early-segmentation larvae. Both cAMP and calcium signaling pathways were enriched in the DEGs of the MIP2-treated larvae, and two neuropeptide receptor genes (Spr, Fmrfar) were up-regulated in the MIP2-treated larvae. The activation of the SPR-cAMP pathway by MIP2 was experimentally validated in HEK293T cells. Furthermore, fourteen cilia-related genes, including Tctex1d2, Cfap45, Ift43, Ift74, Ift22, Cav1 and Mns1, etc. exhibited down-regulated expression in the MIP2-treated larvae. Whole-mount in situ hybridization identified two selected ciliary genes, Tctex1d2 and Cfap45, were specially expressed in circumoral ciliary cells of the early-segmentation larvae. Knocking down Tctex1d2 mRNA levels by in vivo RNA interference significantly increased the larval settlement rate. Conclusion Our findings suggest that MIP2 inhibits the function of the cilia-related genes, such as Tctex1d2, through the SPR-cAMP-PKA pathway, thereby inducing larval settlement in U. unicinctus. The study contributes important data to the understanding of neuropeptide regulation in larval settlement. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10228-y.


Background
Most marine benthic invertebrates exhibit planktic larvae in their life cycle [1].Along with these planktic larval development, they undergo settlement to sediment, rock or other substance surfaces, and then complete larval attachment and metamorphosis [2][3][4].The transition of the larvae from a planktic to a benthic mode is universally recognized as a critical phase in their life cycle and plays a fundamental role in maintaining population quantity and community dynamics [5].For commercial important aquatic animals, this phase holds particular significance in breeding by enhancing the rates of larval settlement and metamorphosis.
Larval settlement is a multifaceted process regulated by the interplay of biotic and abiotic factors [6][7][8][9][10].Neuropeptides, as signaling molecules, have been reported to play a pivotal role in larval settlement and metamorphosis [11][12][13][14][15]. Myoinhibitory peptide (MIP)/allatostatin-B, belonging to the ancient W-amide neuropeptide superfamily, has been demonstrated to be involved in the larval settlement process of certain marine benthic invertebrates.In the annelid Platynereis dumerilii, exogenous application of neuropeptide MIP can prolong the closure time of cilia and induce larval settlement [16].In the barnacle Balanus amphitrite, the mRNA expression pattern of Mip aligns with the larval settlement behavior, suggesting that MIP involves in the settlement process [17].Additionally, MIP plays a vital role in regulating muscle contraction, hormone synthesis, metamorphosis, and ecdysis in arthropods [18][19][20][21][22][23][24][25].SPR (Sex peptide receptor), originally identified as a sex peptide receptor regulating female reproductive behavior in Drosophila melanogaster [26,27], is part of the MIP-SPR pathway, which has been identified to act on sleep homeostasis in D. melanogaster [28].In the nematode Caenorhabditis elegans, MIP signaling through SPRR-2 (sex-peptide-receptor-related-2) modulates salt avoidance learning parallel to the insulin pathway [29].Conzelmann et al. [16] demonstrated that MIP is expressed in chemosensory-neurosecretory cells of the apical organ in P. dumerilii and regulates larval settlement through signal transduction by SPR.Schmidt et al. [30] found that different MIP mature peptides derived from MIP precursors exhibit varying affinities with the receptor, resulting in different signal transmission activities.However, it remains unclear which downstream genes are regulated by the MIP-receptors pathway in inducing larval settlement.
The echiuran worm Urechis unicinctus, a commercially and ecologically important marine benthic invertebrate inhabiting in the intertidal zone, undergoes typical larval settlement and metamorphosis during its life cycle [31].Due to its high nutritional and medicinal values, U. unicinctus is a prized resource in Asia [32,33].Hou et al. [34], through transcriptome analysis, revealed the involvement of the Mip precursor gene in the larval settlement and metamorphosis in U. unicinctus.Lu et al. [35] demonstrated that MIP1 can significantly trigger the settlement of U. unicinctus early-segmentation larvae.Bai et al. [36] identified two MIP receptors, MGIC (myoinhibitory peptide-gated ion channel) and SPR in U. unicinctus, and verified that U. unicinctus MIP1 can activate the SPR-cAMP pathway in HEK293T cells.In this study, we identified the principal MIP inducing earlysegmentation larval settlement in U. unicinctus, and subsequently screened key downstream genes and pathways based on transcriptome and RT-qPCR analysis.Finally, we revealed the spatio-temporal expression characteristics of the cilia-related genes and verified the role of the ciliary gene Tctex1d2 in larval settlement.Our work provides valuable scientific insights into the molecular mechanism of neuropeptides regulating larval settlement and the potential application of MIP inducing larval settlement in U. unicinctus.

Assay of larval settlement treated by MIP mature peptides or PKA inhibitor
The MIP precursor protein of U. unicinctus consists of 475 amino acids with an approximate molecular weight of 60 KDa [34].Thirteen MIP mature peptide sequences (Additional file 1) were predicted from a single gene (GenBank: MT162087.1),and the active MIPs with amidation in the carboxyl terminal of the last amino acid were synthesized by Sangon Biotechnology (Shanghai, China).In accordance with the methodology established by Lu et al. [35], the early-segmentation larvae (12 dpf ) swimming in the upper water layer were collected and transferred to the forty-two glass tubes (diameter = 1.5 cm) with 11.5 cm height of solution, and the spatial distribution of larvae in the water layer was videoed continuously for 5 min.Each tube contained thirty larvae, and the solution in the tubes consisted of filtered seawater (FSW) with 10 µM MIP mature peptides in the experimental groups or the FSW without MIP in the control group.Three replicates were set for each group.The height (H) of each larva in the tube was measured, and the relative height (RH) of larvae was calculated using the formula RH = (H/11.5)%.Larval distribution in each group was quantified, and the MIP with maximum effect on inducing larval settlement was identified as the principal MIP mature peptide.Subsequently, the optimal concentration of the principal MIP was determined using the above experiment system with three concentrations (5, 10, 15 µM) of the principal MIP, respectively.
The PKA (protein kinase A) inhibitor experiment was carried out in six glass tubes (diameter = 2.5 cm) with a height of 10 cm of FSW.Each tube contained 200 earlysegmentation larvae.The larvae in the experiment group were treated with FSW containing 0.5 µM H89, a PKA inhibitor (MedChemExpress, Princeton, USA), while the control group larvae were treated with FSW without the inhibitor.Three replicates were set for each group.After H89 treatment for 12 h, the proportion of larvae settling to the bottom of the glass tube was calculated.Meantime, the larvae were sampled and immediately frozen in liquid nitrogen before stored at − 80℃.

Sampling and total RNA extraction of MIP2-treated larvae
The early-segmentation larvae were utilized for the experiment conducted in glass tubes (diameter = 2.5 cm) with a solution height of 11.5 cm.The solution of the treatment group was FSW with the optimal concentration (10 µM) of the principal MIP (MIP2), while that of the control group was FSW without MIP2.Each tube contained 200 larvae, and there were 3 replicates for each group.The larvae were collected at 1 min, 3 and 5 min of the experiment using 300-mesh sieves, respectively.After the solution containing MIP2 had been filtered, larvae on the sieve were quickly washed with FSW and transferred to the 1.5 mL RNase-free centrifuge tube using a pasteurized straw.Thereafter, the larvae were quickly centrifuged with a small centrifuge for 2-3 s, the supernatant was removed with a pasteurized straw, and then quickly frozen in liquid nitrogen.The sampling and frozen process can be completed within 10-15 s.Finally, the samples were stored at − 80℃.
Total RNAs were extracted from the stored larvae using Sparkzol Reagent (Sparkjade, Jinan, China) according to the manufacturer's instructions.Total RNAs were assessed for amount and integrity using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1% agar gel electrophoresis, and then stored at − 80℃.

Transcriptome analysis
Total RNAs from the larvae treated for 3 min in 10 µM MIP2 and the control groups were used for transcriptome analysis.The mRNA was isolated from the total RNA (1 µg per sample) using oligo-dT beads (Qiagen, Hilden, Germany), and sequencing libraries were constructed using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Ipswich, USA) following the manufacturer's protocols.The libraries (a total of 6 libraries from the two groups with 3 sample replicates) were respectively sequenced using Illumina NovaSeq 6000 (Illumina, San Diego, USA) by Novogene Company (Beijing, China) after their quality was assessed by Agilent 2100 bioanalyzer.
High-quality sequences (clean reads) were obtained from raw reads after eliminating the reads containing ambiguous bases, adapter sequences, etc. Trinity v2.6.6 was employed to jointly assemble all clean reads from the six libraries into unigenes [38].BUSCO was used to assess the completeness of the transcriptome assembly [39].Unigenes were annotated using NT, NR, KO, Pfam, Swis-sProt, GO, and KOG databases.GO annotation was conducted using Blast2GO v2.5 , and functional classification was performed by WEGO [40].
The clean reads were mapped to the assembled whole transcriptome using software RSEM v1.2.15 [41].The expression level of unigenes was quantified with TPM (Transcripts per million) values [42].Differentially expressed genes (DEGs) were analyzed using DESeq2 with the threshold FDR (False Discovery Rate) < 0.05 and|log 2 Fold Change| > 1 [43].GO function enrichment of DEGs was analyzed with GOseq v1.10.0 software.KEGG pathway enrichment analysis of the DEGs was conducted using KOBAS [44].The Benjamin and Hochberg methods were employed to adjust the enrichment p-value, and significance was set with the adjusted p < 0.05.

Plasmid construction and CRE luciferase activity assay
To elucidate the signal transduction mechanism of MIP2, U. unicinctus SPR was expressed in the HEK293T cells by co-transfection of a cyclic adenosine monophosphate (cAMP) response element-luciferase (CRE-Luc) reporter designed to target the cAMP pathway.The open reading frame (ORF) of Spr was isolated by PCR (Additional file 2) and subcloned into the expression vector pcDNA-3.1 (+) to generate the SPR expression vector.Using the expression vector, the HEK293T cells with stable expression of SPR was established.
The HEK293T cells were seeded in 24-well plates at a density of approximately 1 × 10 5 cells/mL per well, followed by an overnight incubation for recuperation.Transfection was performed using Lipofectamine 2000 in Opti-MEM with pcDNA 3.1(+)-Spr, CRE-Luc, and a Renilla luciferase-expression vector, pRL-TK.The total amount of plasmids used in each co-transfection was 300 ng.Subsequently, the cells underwent a 36-h incubation at 37℃ in Dulbecco's modified Eagle medium (DMEN) supplemented with 10% fetal bovine serum (FBS).The cells were maintained in FBS-free DMEM for a 16 h starvation followed by Forskolin (10 µM) (Yeasen, Shanghai, China) and MIP2 (10-10000 nM) treatment for an additional 6 h.Each treatment was set three replicates.Forskolin is an adenylate cyclase activator that directly interacts with the catalytic subunit of the enzyme to increase intracellular cAMP levels [45].
After a quick rinse with ice-cold phosphate-buffered saline (PBS), the cells were dissolved in a passive lysis buffer (Promega, Madison, USA).The resultant cell lysate was used to measure firefly luciferase and Renilla luciferase activities via a Dual-Glo luciferase assay kit (Yeasen, Shanghai, China).Transfection experiments were conducted in quadruplicate with cells cultured in individual wells.

Real-time quantitative PCR (RT-qPCR)
Total RNAs were isolated from the stored samples following the procedure mentioned above.Subsequent to the removal of contaminant genomic DNA using 5 × gDNA digester Mix, the first strand cDNA was reversely transcribed using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) from Yeasen, Shanghai, China.The cDNA sequences of target genes, including Tctex1d2, Cfap45, Ift43, Ift74, Ift22, Cav1 and Mns1 were obtained from U. unicinctus larval transcriptome.Fragments of these genes were respectively amplified with specific primers (Additional file 3), and a 123-bp fragment of U. unicinctus Atpase was served as an internal control gene [46].RT-qPCR was conducted utilizing Hieff UNICON® Universal Blue SYBR Green Master Mix (Yeasen, Shanghai, China) on a qTOWER 3 G Real-Time PCR Thermal Cycler (Analytikjena, Jena, Germany).All reactions were carried out with three sample replicates and three technical replicates.Data were analyzed using the qPCRsoft v4.1 (Analytikjena, Jena, Germany), and the relative expression level of target mRNA was calculated using the 2 −ΔΔCt method.

Whole-mount in situ hybridization (WISH)
Specific primers (Additional file 4) containing Sp6 or T7 promoter sequences were designed according to the cDNA sequences of U. unicinctus Tctex1d2 and Cfap45.Digoxin labeled RNA probes of Sp6-sense and T7-antisense were synthesized using the DIG RNA Labeling Kit Sp6/T7 (Roche, Basel, Switzerland).WISH was conducted following the methodology outlined by Wei et al. [37].Briefly, the samples were digested at 37℃ with 100 ng/mL proteinase K for 15 min (embryos), 20 min (trochophores) or 200 ng/mL proteinase K for 20 min (segmentation larvae), 15 min (worm-shaped larvae).Hybridization was carried out for 16 h at 60℃ using a probe concentration of 1 ng/µL in the hybridization buffer.The samples were incubated with the Anti-Digoxigenin-AP conjugate (Roche, Basel, Switzerland) at a 1: 2500 dilution for 16 h at 4℃, and stained in NBT/BCIP staining solution (Roche, Basel, Switzerland) in the dark for 0.5-1.5 h at room temperature.The results were observed and photographed using a Leica DM2500 LED microscope (Leica, Weztlar, Germany).Drawings and final panels were designed using Adobe Photoshop CS6 (San Jose, CA, USA).

RNA interference (RNAi)
The cDNA fragments of Tctex1d2 and EGFP (enhanced green fluorescent protein gene) were generated using the primers tagged with T7 sequence for dsRNA synthesis (Additional file 5).The quality of the PCR products was confirmed by 1% agar gel electrophoresis, followed by purification with the MolPure® PCR Purification Kit (Yeasen, Shanghai, China).The Tctex1d2-dsRNA and EGFP-dsRNA (as negative control) were synthesized using the MEGAscript® RNAi kit (Invitrogen, Waltham, USA) following the manufacturer's protocols.The dsR-NAs were assessed for quality and quantity using a Nano-Drop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and 1% agar gel electrophoresis, and then stored at − 30℃ until use.
The larvae were reared following the method described by Wei et al. [37].The early-segmentation larvae (10 dpf ) were randomly divided into three groups: the Tctex1d2-dsRNA group, EGFP-dsRNA group, and blank control group.Each group had three replicates, with 170 larvae in each replicate.The larvae of the Tctex1d2-dsRNA group and EGFP-dsRNA group were initially soaked in 3 mL FSW with a final dsRNA concentration of 50 nmol/L for 12 h, and then respectively transferred to glass tubes containing 50 mL FSW without dsRNA for 60 h.For the blank control group, the larvae were treated in 3 mL FSW without any dsRNA, and other conditions were the same as the dsRNA treatment groups.At the end of larval treatment, number of the settled larvae was recorded.The settled larvae were defined as those attaching to the bottom surface of the glass tubes and unable to swim into the seawater layer.The settlement rate was calculated using the formula: the number of settled larvae / the total number of larvae * 100%.
To assess the efficiency of gene knockdown, the larvae were collected at 72 h post dsRNA treatment and stored at − 80℃ after immediately frozen in liquid nitrogen.Total RNAs from the nine samples across the three groups were respectively extracted using Trizol reagent (Yeasen, Shanghai, China).The cDNA synthesis was performed and the expression level of Tctex1d2 in the larvae was determined by RT-qPCR to assess the RNAi effect.

Statistical analysis
All data were presented as mean ± SEM.One-way analysis of variance (ANOVA) followed by Duncan's multiplerange test and independent samples T-test, using SPSS v22 software (SPSS Inc., Chicago, USA) was employed to determine significant differences between means.Statistical significance was set at p < 0.05.

MIP2 as the principal MIP mature peptide in triggering larval settlement of U. unicinctus
We synthesized 13 MIP mature peptides and detected the effect of these MIPs on triggering the early-segmentation larval settlement (Additional file 6 and 7).The results showed that 12 MIPs (MIP1-11 and MIP13) significantly induce the larval settlement, with the exception for MIP12 (Fig. 1A-C).In MIP2 treatment group, the average relative height of larvae in the water layer was always the lowest, exhibiting reduction of 18.80% (1 min), 32.34% (3 min) and 46.18% (5 min) in comparison to that of the control group, respectively.Following closely was MIP5, which displayed reductions of 18.42% (1 min), 29.24% (3 min) and 43.55% (5 min) relative to the control group, respectively.Moreover, the percentage of larvae in the lower water layer (0-5 cm) was the largest in MIP2 group at the 5 min treatment, constituting 42.22% (Fig. 1D and Additional file 8).Based on the relative height of the larvae in the water layer and the amount of the larvae in the lower water layer, MIP2 emerged as the principal MIP mature peptide in triggering larval settlement of U. unicinctus.Up subjecting the early-segmentation larvae to varying concentrations of MIP2, the larval relative height (RH) in the 10 or 15 µM MIP2 groups was significantly lower than that of 5 µM MIP2 group and control group.Notably, no significant difference was observed between the 10 µM MIP2 and 15 µM MIP2 groups (Fig. 1E).Consequently, the optimal concentration for MIP2-induced early-segmentation larval settlement was determined to be 10 µM based on the lowest RH value.Intriguingly, we observed that after 7 h of MIP2 treatment, the circumoral cilia of the larvae underwent closure, in stark contrast to the dispersed arrangement observed in the control larvae (Additional file 9).

Identification and enrichment analysis of DEGs in transcriptomes between MIP2-treated and control larvae of U. unicinctus
Six cDNA libraries were constructed from both the control and MIP2-treated larvae, yielding a total of 132.82 Mb raw reads, and subsequent processing resulted in 131.76 Mb clean reads.The assembly produced 43,034 unigenes with an average length of 1476 bp and N50 of 2581 bp (Tables 1 and 2).The RNA-seq data have been deposited in the NCBI database with the accession number of PRJNA1027755.
A comprehensive analysis revealed 7519 DEGs, comprising 4940 up-regulated and 2579 down-regulated genes in MIP2-treated larvae (Fig. 2).Notably, several genes associated with neuropeptide receptor or key signaling molecules (Additional file 10) and fourteen cilia-related genes (Additional file 11) were contained in the down-regulated DEGs.GO enrichment analysis of the down-regulated DEGs identified eleven significantly enriched terms, such as structural constituent of ribosome, organelle, and protein-containing complex (Fig. 3A).Conversely, only three GO terms, namely cell adhesion, extracellular region and peptidase activity were significantly enriched in the up-regulated DEGs (Fig. 3B).
KEGG enrichment analysis showed that the downregulated and the up-regulated genes were enriched in 241 and 327 signaling pathways, respectively (Additional file 12 and 13).Among the down-regulated DEGs, ribosome was the sole significantly enriched signaling pathway (Fig. 3C), while the up-regulated DEGs featured nine enriched pathways, including protein digestion and absorption (Fig. 3D).Interestingly, several pathways were concurrently enriched in both down-regulated DEGs and up-regulated DEGs, such as cAMP, MAPK, AMPK, Notch, Rap1 and calcium signaling pathways (Additional file 12 and 13).

MIP2 activates SPR in a dose-dependent manner
We found that the expression of the neuropeptide receptor gene Spr was significantly increased in MIP2-treated larvae, and previous studies have demonstrated that U. unicinctus MIP1 can activate SPR [36].To examine the effects of MIP2 on the receptor SPR, HEK293T cells were employed, wherein Spr-ORF was co-transfected with the CRE-Luc reporter system to evaluate the potential MIP2 signaling pathway coupled to SPR receptor in U. unicinctus.The result showed that U. unicinctus MIP2 exhibited inhibitory effects on Forskolin-stimulated CRE-Luc activity in a dose-dependent manner in HEK293T cells (Fig. 4).

Expression characteristics of ciliary genes and settlement rate of the early-segmentation larvae treated by MIP2 and H89
Seven cilia-related genes were selected from the above fourteen genes, and their expression dynamics in earlysegmentation larvae treated with MIP2 were scrutinized using RT-qPCR.Three expression patterns emerged among these genes.Specifically, the mRNA levels of the four genes, Tctex1d2, Ift74, Cav1 and Ift22 were consistently down-regulated significantly at 1 min, 3 and 5 min post MIP2 treatment compared to that of the control group.In contrast, Cfap45 and Mns1 demonstrated significantly down-regulated at 1 and 3 min but a pronounced up-regulation at 5 min.Intriguingly, Ift43 exhibited significant down-regulation only at 3 min, but noteworthy up-regulation at 1 and 5 min (Fig. 5).The results of RT-qPCR were consistent with that of the  transcriptome, suggesting that responses of these genes to MIP2 are distinctly different in both intensity and timing.H89 is the cAMP-dependent PKA inhibitor, which regulates the expression of downstream genes by inhibiting the PKA activity to reduce the phosphorylation of target proteins.After H89 treated, the larval settlement rate increased significantly by 21.7% compared to the control group (Fig. 6A).The results of RT-qPCR showed that all the above seven cilia-related genes were significantly down-regulated in the early-segmentation larvae after H89 treatment (Fig. 6B).

Temporal and spatial expressions of Tctex1d2 and Cfap45 in U. unicinctus embryos and larvae
Two representative genes, Tctex1d2 and Cfap45, exhibiting disparate response patterns to MIP2, were selected for a detailed exploration of their expression patterns in U. unicinctus embryos and larvae.FPKM values retrieved from transcriptome data (NCBI accession number: PRJNA485379) revealed nuanced patterns.Tctex1d2 exhibited a gradual increase from 2 to 8 cell embryo to trochophore, followed by a decline from late-trochophore to worm-shaped larva (Additional file 14).In contrast, Cfap45 demonstrated its highest expression in 2-8 cell embryo, diminishing significantly in multicellular embryo, blastula and gastrula.Notably, Cfap45 expression spiked in trochophore, and gradually decreased from late-trochophore to worm-shaped larva (Additional file 14).
The WISH results further validated these expression patterns.Obvious positive signals of Tctex1d2 mRNA initially appeared in the gastrula, displaying a dispersed distribution (Fig. 7A a-f ).After hatching, the strong positive signals of Tctex1d2 mRNA were mainly located in the circumoral ciliary cells of trochophores or segmentation larvae (Fig. 7A g-k).In the worm-shaped larva, the Tctex1d2 positive signals were observed in the digestive tract and the inner side of the body wall (Fig. 7A l).Positive signals of Cfap45 mRNA were diffuse in the embryos at the different stages, with the strongest signal observed in zygote (Fig. 7B a'-f ').In the trochophore and segmentation larvae, the positive signals were predominantly located in the circumoral ciliary cells and the apical tuft cells of early-trochophore larvae (Fig. 7B g'-k').Similar to Tctex1d2, in the larva, Cfap45 mRNA signals were also observed in the digestive tract and the inner body wall (Fig. 7B l').No positive signal was detected in embryos and larvae treated with Tctex1d2 or Cfap45 negative probes (Additional file 15).

Tctex1d2 mRNA knockdown promotes settlement of the early-segmentation larvae
RT-qPCR analyses demonstrated a significant reduction (p < 0.001) in the expression level of the ciliary gene Tctex1d2 in the Tctex1d2-dsRNA group after RNAi for 72 h, representing a 43.76% decrease compared to the blank control group (Fig. 8A).Importantly, the settlement rate of larvae in the Tctex1d2-dsRNA group increased extremely significantly, reaching 57.65% (48 h after RNAi) and 69.19% (72 h after RNAi), respectively.In contrast, the settlement rates were only 10.06% (48 h) and 15.09% (72 h) in the blank control group, and 14.16% (48 h) and 18.40% (72 h) in the EGFP-dsRNA group (Fig. 8B-C).These findings underscore the significant impact of Tctex1d2 knockdown in promoting the settlement of early-segmentation larvae.

The effect of MIPs on larval settlement in U. unicinctus correlates with receptor affinity
The process of larval settlement is intricately regulated by diverse internal and external signals, exerting a profound influence on the normal development and survival of animals.Neuropeptides have been recognized as a critical role in this intricate process [12,14,47,48].Maturation of neuropeptides involves the cleaving of neuropeptide precursors, with various mature peptides from the same precursor exhibiting distinct functions [30,49].MIP has been revealed in promoting larval settlement in several marine invertebrates.For example, MIP1 and MIP4-MIP7 in P. dumerilii significantly induce the downward vertical movement of trochophore at the concentration of 5 µM [16].In Tenebrio molitor, MIP5 stimulates contractile activity in beetle oviduct muscles at a concentration of 10 nM [50].In U. unicinctus, we revealed that 12 MIP mature peptides, excluding MIP12, significantly induce the early-segmentation larval settlement.Among them, MIP2 emerged as the principal MIP, with the most effective dose being 10 µM (Fig. 1E).This suggests the similar characteristics, despite variations in the MIP concentration among the different species [16].
Schmidt et al. [30] observed variations in the affinity of 11 MIPs to their receptors, MAG (myoinhibitory peptide activated GPCR) and MGIC in P. dumerilii.MIP6, for example, activates MAG with EC 50 values in the nanomolar range, being the most potent ligand.In contrast, MIP2 is a less effective ligand, activating MAG at micromolar concentration.Notably, for MIGC, MIP2 emerges as the most potent ligand.This divergence in the effects of different MIP mature peptides in U. unicinctus may stem from variations in receptor affinity.This, in turn, may lead to the activation of associated signaling pathways, resulting in the differences in the effects on downstream target genes.Consistently, in both P. dumerilii and U. unicinctus, MIP treatment triggered larval settlement and induced alterations in larval ciliary behavior (Additional file 9), underscoring the universality of MIP in inducing larval settlement of marine benthic invertebrates.

Multiple signaling pathways involved in MIP2-induced larval settlement
In this study, a total of 7519 DEGs (17.47% of all unigenes) were identified in the transcriptomes of larvae subjected to the MIP2 treatment compared to the control group.Subsequently, a multitude of signaling pathways exhibited enrichment in both down-regulated and upregulated DEGs (Additional file 12 and 13).Larval settlement caused by short-term neuropeptide treatment is prevalent in marine benthic invertebrates [16,35,47], and larval settlement is a complex process, mediated by diverse signaling pathways [51].Furthermore, changes in phenotype and numerous gene expression caused by the treatment of exogenous stimuli within minutes have been reported in other species [52][53][54].Although the mechanism of rapid biological response to this exogenous stimulus has not been fully elucidated.
Neuropeptides conventionally engage the G-proteincoupled receptor signaling pathway.Conzelmann et al. [16] proposed that MIP7 induces prolonged and frequent ciliary arrests in P. dumerilii trochophore, utilizing the conserved G-proteincoupled receptor signaling pathway to regulate the larval settlement.Additionally, Bai et al. [36] verified the capability of U. unicinctus MIP1 to activate its SPR receptor, thereby reducing the intracellular cAMP concentration in HEK293T cells.Our findings revealed that MIP2 induced a decline in intracellular cAMP concentration by activating the SPR receptor, as evidenced by the use of a CRE luciferase reporter plasmid.It is noteworthy that ADCY3 (adenylate cyclase 3) mediates ATP to cAMP conversion [55], and cAMP as a second messenger activates PKA pathway by stimulating PKA catalytic activity.Conversely, PDE4 (phosphodiesterase 4) hydrolyzes cAMP [56].Consequently, the observed decrease in Adcy3 expression or an increase in Pde4 expression following MIP2 treatment signified a reduction in cAMP concentration (Additional file 16), thereby inhibiting the PKA activity.These findings strongly supported the assertion that MIP2 induces the larval settlement by the SPR-cAMP pathway.
When treating the early-segmentation larvae with the PKA inhibitor (H89), we observed a significant increase in larval settlement rate and a significant reduction in the expression levels of ciliary genes (Fig. 6).This aligned with the results following MIP2 treatment, suggesting that MIP2 regulates downstream genes by inhibiting PKA, thus delineating the SPR-cAMP-PKA pathway.It has been reported that peptides bind to GPCRs to activate phospholipase C (PLC), cause changes in Ca 2+ concentration, activate Calmodulin-dependent protein kinase (CaMK), and thus regulate downstream effector genes [57,58].In this study, a significant elevation in mRNA level of the neuropeptide FMRFamide receptor gene (Fmrfar) and calcium signaling pathway related genes (Calm, Camta1, Camkk1, CamkII and Plcb4) was noted post MIP2 treatment.Given that the neuropeptide FMRFamide receptor has been associated with stimulating Ca 2+ signals and modulating the activity of dopaminergic neurons in D. melanogaster, and that CaMKII serves as a downstream signaling component of FMRFamide receptor [59].Therefore, we speculated that MIP2 holds the potential to activate the FMRFamide receptor.This activation triggers PLC in turn, expediting the decomposition of PIP2 (phosphatidylinositol(4,5)  bisphosphate), resulting in the release of Ca 2+ from the endoplasmic reticulum and an increase in the expression of the calmodulin gene Calm, then CaMKII is further activated to regulate the expression of downstream ciliary genes.
Conzelmann et al. [47] postulated the thrust exerted on the body is proportional to the beat frequency of cilia during larval swimming, where alternating phases of spontaneous beating and closure of cilia govern swimming depth in planktonic larvae.The coordinated beating of cilia in eukaryotes is regulated by axonemal heavy chain dynein ATPases, which are motor proteins generating force along microtubules through ATP hydrolysis [60].Our results elucidate KEGG terms mainly involved in energy metabolism, encompassing protein digestion and absorption, glycerophospholipid metabolism, carbohydrate digestion and absorption, pentose and glucuronate interconversions, linoleic acid metabolism, arachidonic acid metabolism, glycolysis/gluconeogenesis, fat digestion and absorption, fatty acid degradation, fructose and mannose metabolism signaling pathways (Additional file 12 and 13).This metabolic pattern mirrors observations in larval settlement progress of other marine invertebrates, such as the barnacle B. amphitrite [61][62][63][64] and Fujian oyster Crassostrea angulate [65].Moreover, we also noticed that enrichment in several signaling pathways (MAPK, AMPK, Notch, Rap1 and Foxo) associated with signal transduction (Additional file 12 and 13), known for their conservation in participating in larval settlement of marine invertebrates [66][67][68][69].AMPK, a cellular energy sensor pivotal in maintaining the energy balance of the cell and the whole body [70], and FoxO, involved in crucial cellular processes like metabolism, apoptosis, proliferation and survival, may potentially play a role in MIP2-induced larval settlement of U. unicinctus.The adhesion process, crucial for survival and settlement in sessile organisms, was also implicated in our study.Focal-adhesion signaling pathway, including genes RHO and RHO1, and ECM-receptor interaction, were significantly enriched (p < 0.05), suggesting the involvement of adhesion alterations in U. unicinctus larval settlement.
In conclusion, the induction of larval settlement in U. unicinctus by MIP2 is a complex physiological phenomenon, involving many interconnected biological processes spanning organismal systems, metabolism and environmental information processing, among others.

MIP promotes larval settlement by regulating the ciliary gene expression
Cilia, ubiquitous organelle presenting on the larval surface of various marine invertebrates, play a crucial role in facilitating larval swimming and aiding in feeding processes [71].In U. unicinctus, cilia were initially observed as uniform distributed short structures on the body surface in the blastula under light microscope (Additional file 17: Fig. S7E-F).Subsequently, in the early-trochophore, cilia on the body surface manifested mainly as an apical ciliary tuft, circumoral ciliary ring and telotroch.In the mid-trochophore, the apical ciliary tuft disappears (Additional file 17: Fig. S7G-H).After that, in late-trochophore, early-segmentation larva and segmentation larva, cilia were mainly in the form of circumoral ciliary ring and telotroch (Additional file 17: Fig. S7I-K).Eventually, in the worm-shaped larva, all cilia on the body surface disappeared completely (Additional file 17: Fig. S7L) [72].The settlement process will be initiated normally during the early-segmentation larva in U. unicinctus (Additional file 17: Fig. S7J), but this natural settlement was extremely slow and usually took 2-3 days from the water surface to the bottom.While, there was significant settlement in the MIP2-treated earlysegmentation larvae (Fig. 1A-C).The activity of cilia is responsive to environmental cues and is generally regulated by nervous system [73][74][75].Neuropeptides known to regulate larval settlement by influencing ciliary activity have been documented in several species, including P. dumerilii [46], Crepidula fornicate [76], T. transversa [77], and Lineus longissimus [78].Although the diversity in effects (activation or inhibition) of different neuropeptides on ciliary beat frequency, it remains unclear which cilia-related genes are specifically regulated during the process.
In this study, utilizing the transcriptome data from the larvae in both the MIP2 treated and control groups, we identified fourteen previously reported cilia-related genes (Additional file 11).These genes mainly function in ciliogenesis/assembly and ciliary beating (Table 3).Furthermore, we identified the predominant localization of mRNA for two selected ciliary genes, Tctex1d2 and Cfap45, in circumoral ciliary cells of U. unicinctus early-segmentation larvae by WISH (Fig. 7).Additionally, SPR, a MIP receptor which was expressed in the ciliary zone of larvae [36] was also identified to increased significantly in MIP2-treated larvae (Additional file 10), indicating that MIP2 may regulate ciliary gene expression through SPR.Knocking down the mRNA level of Tctex1d2 by RNAi resulted in a significantly increased for the larval settlement rates, by 3.59 times at 48 h and 4.73 times at 72 h compared to the blank control group (Fig. 8).TCTEX1D2, recognized as a light chain of the dynein-2 complex, collaborates with the IFT-A complex to facilitate retrograde ciliary protein transport [96].Perturbing the Tctex1d2 gene, either through knockout or mutation, typically reads to phenotypes such as short cilia [97], delayed ciliogenesis [98], or a reduction in ciliated cells [99] in mammalian cells and zebrafish.CFAP45 (Cilia-and flagella-associated protein 45) supports ciliary and flagellar beating in mammalian through an adenine nucleotide homeostasis module.In Cfap45-deficient embryos and cells, the ciliary beat frequency [60] or the ciliary stability [79] is reduced.Consequently, we propose that the ciliary genes identified in this study, especially Tctex1d2 and Cfap45, play crucial roles in the larval settlement, and MIP2 promotes U. unicincus larval settlement by regulating the expression of these ciliary genes.

Conclusions
Our study establishes that MIP2 serves as the principal neuropeptide inducing the larval settlement process in U. unicinctus by the SPR-cAMP-PKA pathway, thereby regulating the expression of pivotal downstream ciliary genes, including the key gene Tctex1d2.Notably, our investigation highlights the substantial involvement of energy metabolism and signal transduction pathways in the MIP2-induced larval settlement of U. unicinctus.To our knowledge, this research represents the inaugural documentation of the action pathway through which the neuropeptide MIP induces the larval settlement in U. unicinctus.The insights garnered from our findings are poised to significantly contribute to future research endeavors focused on unraveling the intricacies of neuropeptide regulation in larval settlement process.

Fig. 1
Fig. 1 MIP-induced larval settlement assays.A-C: Relative height of the larvae treated with 13 MIPs at 1 min, 3 and 5 min; D: Percentage of the larvae in 0-5 cm water layer after treated by 13 MIPs for 5 min, respectively; E: Relative height of the larvae treated by MIP2 at the different concentrations for 5 min.Each rhombus represents a data.All data are represented as the mean ± SEM from three biological replicates.Different letters indicate significant differences (p < 0.05)

Fig. 2
Fig. 2 Volcanoplot illustrating the differentially expression of unigenes between the control group larvae and those treated with MIP2 for 3 min.Pink and green spots indicate the differentially expressed unigenes, while blue spots indicate no differentially expression

Fig. 3
Fig. 3 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes (DEGs).A: GO enrichment analysis of the down-regulated DEGs; B: GO enrichment analysis of the up-regulated DEGs; C: KEGG enrichment analysis of the downregulated DEGs; D: KEGG enrichment analysis of the up-regulated DEGs

Fig. 6 Fig. 5
Fig.6 Settlement rate and target gene mRNA relative levels of the larvae treated with H89.A: Settlement rate of the larvae treated with H89.B: Relative mRNA level of seven ciliary genes in the larvae treated with H89.Data are represented as the mean ± SEM from three biological replicates.Asterisks indicate significant differences between H89 groups and the control group (* p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 8
Fig. 8 Gene knockdown efficiency and the settlement rate of the larvae after RNAi.A: Gene knockdown efficiency of Tctex1d2 in dsRNA-treated larvae for 72 h.B: Larval settlement rate at 48 h of RNAi.C: Larval settlement rate at 72 h of RNAi.Data are represented as the mean ± SEM from three biological replicates.Asterisks indicate significant differences between Tctex1d2-dsRNA group and blank control group (*** p < 0.001)."ns" indicates no significant differences between EGFP-dsRNA groups and blank control group

Table 1
Overview of sequencing reads from six larval transcriptomes of U. unicinctus

Table 2
Length distribution of the assembled transcripts and unigenes in U. unicinctus larval transcriptomes